1. Field of the Invention
The present invention relates generally to the fields of immunology and protein chemistry. More specifically, the present invention relates to oncofetal antigen specific T-lymphocyte subclass mediated immune responses: manipulation and uses of oncofetal antigen specific CD4, CD8 cytotoxic and suppressor T cells and interleukin-10 for early cancer detection tests, for conventional therapy monitoring, and for immune-intervention through autologous T-cell therapy and anti-cancer vaccination.
2. Description of the Related Art
Established tumors can grow and kill the host bearing such tumors even though lymphocytes obtained from that host animal can adoptively transfer tumor immunity to other syngeneic animals (1-3). Also, investigators have shown that a tumor-bearing animal can reject challenge with part of that tumor when inoculated with tumor cells at a different site on its body (1, 4). This phenomenon has been termed concomitant immunity (2-4). Tumors can evade tumor-reactive lymphocyte-mediated destruction b y inhibiting protective immune responses directly, by secretion of inhibitory cytokines, and indirectly, by activating inhibitory regulatory elements of the immune system (5-11).
It has been suggested that rodents, like humans, challenged with carcinogens such as DNA-altering chemicals, radiation or oncogenic viruses respond as either xe2x80x9cprogressorsxe2x80x9d, which develop advanced lethal tumors and die or xe2x80x9cregressorsxe2x80x9d, which fail to develop the fully malignant tumor cells giving rise to cancer. The xe2x80x9cregressorsxe2x80x9d immunologically manage to control the tumor""s growth or existence. These protective immune mechanisms at work in the regressors are widely believed to occur through cell-mediated responses mediated by a T-cell subclass, termed CD8 T-Cytotoxic or TC cells, and/or assisted by other antigen-specific T-lymphocyte subclasses including CD4 T-Helper 1 or TH-1 subclasses.
Approximately 60% of RFM mice develop lethal thymic lymphomas during a six month period subsequent to fractionated, sub-lethal X-irradiation (12,13). Systematic sampling of thymocytes of the irradiated mice during the first six months post-irradiation using intrathymic challenge assay into normal syngeneic mice revealed that when any OFA+ thymocytes were transferred to normal thymus, a high correlation of adoptive induction of T-cell lymphoma was observed, suggesting that oncogenic cells were induced in all irradiated recipients by six months. However, only approximately half of the irradiated donor mice developed lymphomas. The irradiated mice that survive the first 6 months never show any physical signs of tumor development.
It has been shown that mice which had been irradiated 11 months previously and appeared tumor-free, had developed clonable memory CD4 and CD8 effector T cells which were specific for a 44 kDa oncofetal antigen (OFA) (14). It was also determined that age-matched, non-irradiated RFM mice yielded OFA-specific T cell clones; however the frequency of these T cell clones was significantly lower than the frequency in the long-term irradiation survivors, and the non-irradiated mice yielded no clones with high affinity anti-OFA T cell receptors. Immunobiology teaches that animals and humans which retain the capacity to respond to T or B-cell stimulating immunogens retain low affinity precursors and are able to respond to such non-self. Thus it is not surprising that such OFA-reactive memory T cells would be induced in the irradiated mice, since OFA+ thymus cells are detectable as early as 2 weeks after irradiation but are entirely absent from non-irradiated, normal RFM/UnCr mice (13, 15).
However, even with these memory effector T cells that are tumor-reactive, challenge of previously-irradiated mice yielded no increased resistance to RFM lymphoma cells. In fact, such previously-irradiated mice showed significantly enhanced tumor growth kinetics compared to non-irradiated, age-matched controls that were challenged with the same tumor cells (14). This is likely because the previously-irradiated, long-term survivor mice had not only effector T cells, but also CD8+ non-cytototoxic T cells that did not secrete interferon-xcex3. These non-cytotoxic CD8 T cells must secrete some factor(s) which inhibits the cytotoxic activity of anti-OFA cytotoxic T cell clones but does not inhibit Tc clone cell proliferation (14).
All modern summaries of tumor immunobiology from other laboratories, attempting to characterize a host""s immune response to emerging antigenic cancers (e.g., references 48-52), focus on the means by which the primary tumors and metastases xe2x80x9cescapexe2x80x9d the host""s various humoral and cellular-mediated immune responses directed against the tumor. The focus has been instead on unshared, individual tumor specific transplantation antigen (TSTA). Rarely is a shared, host-cell encoded, tumor associated transplantation antigen (TATA) mentioned as the target for the specificity of these immune response. However, the focus of the present invention is on the 44 kD oncofetal antigen (44 kD OFA). OFA is an antigen which is normally expressed in embryonic and fetal tissue as phase-specific, developmentally regulated, embryonic antigen. The experiments leading to the present invention demonstrate, via flow cytometry and binding studies with anti-OFA monoclonal antibodies, that 44 kD OFA is distributed widely on all tumors of rodents and humans as a tumor-specific, but not a tumor subclass-specific, antigen or immunogen (see, e.g., refs 54-67). Since human cancers generally express 44 kD OFA, they too stimulate similar T-cell subclasses. Thus, identification of these anti-OFA responses in humans and animals during the development of cancer is used in the methods of the present invention to detect tumor presence and host mediated T-cell immune responses to emerging cancers using (a) peripheral blood lymphocytes (PBLs) of cancer patients as a source of OFA-specific precursors, (b) allogeneic or xenogeneic 44 kD OFA as a source of 44 kD OFA, and (c) autologous antigen processing cells to process the OFA for stimulation.
The prior art is deficient in effective means for screening individuals for shared oncofetal antigen (OFA) expression during early stage carcinoma and/or leukemia or lymphoma development. In addition, the prior art is deficient in effective means for monitoring a patient""s immune response to oncofetal antigen (OFA) during treatment of the cancer. The present invention fulfills this longstanding need and desire in the art.
The present invention discloses that the inhibitory substance secreted by the non-cytotoxic CD8 T cell clones can inhibit T cell secretion of interferon-xcex3, is not antigen-specific, and is not MHC-restricted. The inhibitory substance, however, is neutralized by anti-IL-10 monoclonal antibody but not by an isotype control antibody. Also, the supernatants of these antigen-restimulated, non-cytotoxic CD8 T cells contain IL-10, while the supernatants of antigen-restimulated, cytotoxic CD8 T cell clones do not. The present invention thus also discloses that inclusion of anti-IL-10 antibody in the cultures of the non-cytotoxic CD8 T cell clones, rescues their anti-tumor cytotoxic ability. Further, it is shown that the IL-10 does not come from macrophages or tumor cells, but from the clones. Macrophages are not the targets of the inhibitory activity, but appear to act on the Tc clone cells. Thus, the present invention demonstrates that CD8 T cells take on the functional activity of xe2x80x9csuppressorxe2x80x9d T cells for cell-mediated immunity by having the gene for IL-10 activated and the secretion of that cytokine can mask the functional potential of the secreting T cell itself.
It has been reported that in irradiated, long-term surviving RFM mice there is enhanced kinetics of tumor development upon challenge with RFM lymphoma cells. Splenic OFA-specific, non-cytotoxic, CD8+ T cells from such mice were cloned. Upon antigen stimulation, these non-cytotoxic CD8+ T cell clones secrete a factor that inhibits the ability of OFA-specific RFM Tc cell clones from killing 5T RFM lymphoma cells in vitro. The supernatants from non-cytotoxic, CD8+ T cells do not inhibit the tumor cell-induced proliferation of the Tc cell clones, however. The present invention demonstrates that OFA-stimulated, non-cytotoxic, CD8 T cell clone culture supernatants also inhibit interferon-xcex3 secretion by stimulated CD4 and CD8 anti-OFA effector T cell clones in a dose-dependent manner. The inhibitor in those culture supernatants acts neither in an antigen-specific nor MHC-restricted manner. OFA-stimulated non-cytotoxic CD8 T cell clones"" culture supernatants contain IL-10, while those from OFA-stimulated, RFM OFA-specific Tc clones do not. Moreover, the monoclonal anti-IL-10 antibody specifically blocks the inhibition of cytotoxic activity and interferon-xcex3 secretion by OFA-specific CD8 and CD4 effector T cell clones in a dose-dependent manner in vitro. Incorporation of anti-IL-10 antibody into the cytotoxicity assays of the OFA-specific, non-cytotoxic CD8+ T cell clones against 5T tumor cells restores their cytotoxic activity.
In one embodiment of the present invention, there is provided a method of determining the success of a cancer therapy in an individual, comprising the step of measuring the amount and frequency of oncofetal antigen-specific T-Cell subsets in the individual""s peripheral blood lymphocytes (PBLs) or in tumor infiltrating lymphocytes (TILS) at the residual tumor site.
In another embodiment of the present invention, there is provided a method of determining whether protective immunity against a tumor will develop in an individual, comprising the step of measuring the frequency of oncofetal antigen-specific T cells which secrete IL-10 at the site of the tumor.
In yet another embodiment of the present invention, there is provided a method of determining the potency of the protective anti-tumor immunity in an individual, and the phenotype and composition of the T-cell subclasses involved, comprising the step of measuring the frequency of interferon-xcex3 secreting T cells and oncofetal antigen-specific T cells at the site of the tumor.
The present invention is drawn to a method of stimulating and causing clonal expansion of memory CD4 helper cells, CD8 Tc cytoxic lymphocytes and CD8 non-cytotoxic T-suppressor lymphocytes comprising administering an effective dose of purified 44 kDa oncofetal antigen. Further, the present invention is drawn to a method for activating T-suppressor cells comprising inhibiting or limiting IL-10 production of said cells.
An additional method provided in the present invention is a method of screening an individual for early stage carcinoma, lymphoma development comprising: cloning oncofetal antigen specific T-cells from said individual; and determining a frequency of cytoxic T-cells and inhibitory T-suppressor cells that cause specific suppression of CD8 and CD4 cytoxicity.
In addition, the present invention provides a method for monitoring succes of cancer therapy and determining whether protective immunity will develop in an individual, comprising the step of measuring a frequency of oncofetal antigen-specific T cell subclasses, including CD8 cytotoxic T-cells and T-cells making IL-10, in said individual, wherein when said frequency of CD8 cytoxic T-cells is high and said frequency of T-cells making IL-10 is low, therapy is successful and development of protective immunity is likely.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.